Publication Abstract

Optimisation and standardisation of PCR assay protocols for koi herpesvirus

S. Hartshorn, G. Woolford, K. Way, D.M. Stone and P.F. Dixon

Koi herspesvirus (KHV) is a recently recognized virus associated with mass mortalities in koi carp (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio). The disease has rapidly spread around the world through international trade in ornamental carp and devastating losses have been seen in carp populations in the United States, Israel, South Africa, Indonesia, Japan, South-east Asia and Europe. The virus has been difficult to detect using traditional culture methods and therefore a range of PCR-based methods are cited in the literature. An international workshop on KHV in 2004 highlighted the need for standardisation of detection methods, and in particular PCR assays, for KHV. In response, a research study was initiated at the Cefas Weymouth laboratory. Published PCR protocols for detection of KHV were compared for sensitivity and specificity with PCR protocols based on protein coding regions of the KHV genome. Then the tissue sampling, extraction and amplification protocols were optimised for the most effective assays. In further studies, primer sets targeting smaller sequences of KHV genome were found to be more reliable in detecting the degraded DNA found in  decomposed tissue samples. From these studies a standardised protocol has been developed and the Bercovier-TK and modified Gray SpH primer sets selected as the most robust for detection of KHV DNA in a range of tissue samples. The protocol has been adopted as the standard at the CEFAS Weymouth laboratory and other laboratories around the world have been contacted to request their participation in a ring-trial to complete the validation of the protocol.

Reference:

S. Hartshorn, G. Woolford, K. Way, D. M. Stone and P. F. Dixon. (Not yet published) Optimisation and standardisation of PCR assay protocols for koi herpesvirus. 5th International Symposium on Aquatic Animal Health, September 2-6, 2006, San Francisco, USA